Objective：This study aims to characterize the gene mutation in myeloid malignancies with normal karyotype by exploring multiple gene mutations. Methods：Tatal 102 acute myelogenous leukemia（AML） with normal karyotype and myelodysplastic syndrome（MDS）paticnts were retrospectively analyzed in Changzhou Second Affiliated Hospital of Nanjing Medical Univcrsity and the First Affiliated Hospital of Suzhou University. Targeted second generation sequencing were performed using a custom-designed 49-gene panel. CALR，FLT3 internal tandem duplication（FLT3-ITD），NPM1 and CEBPA mutation were detected by Sanger sequencing. Results：①Together，gene mutations accounted for a considerable frequency of 98.8% AML patients. Coexistence of ≥ 3 mutations was identified in 52.4% patients. The most commonly mutated gene was NPM1（35.4%），followed by FLT3-ITD（25.6%），CEBPA double mutations（24.4%），DNMT3A（19.5%），TET2（18.3%），NRAS（13.4%），RUNX1（11.0%）and CSF3R（11.0%）mutations. ②The gene mutations were present in 90% MDS patients. Coexistence of ≥ 3 mutations was in 55.0% patients. The most commonly mutated gene was RUNX1（35.0%），followed by ASXL1（25.0%），SF3B1（15.0%），FLT3-TKD（15.0%）and BCOR（15.0%）mutations. ③The incidence of mutation was similar in AML patients and MDS patients（P=0.097）. The mutation rate of each of NPM1 and CEBPA double mutations was higher in patients with AML（P < 0.05），while RUNX1，ASXL，SF3B1 and BCOR mutations were identified more frequently in MDS patients，both differences were significant（P < 0.05）. ④Gene aberrations involved in DNA methylation（DNMT3A，TET2，IDH1/2）and receptors/kinases（FLT3-ITD，FLT3-TKD，JAK1，JAK2，JAK3，c-KIT，PDGFRA，PDGFRB，MPL，CSF3R，NOTCH1，IL7R）significantly predominated in AML while post-translational chromatin modification（EZH2，ASXL1/2，SETD2）and RNA splicing（SRSF2，SF3B1，ZRSR2，U2AF1）significantly predominated in MDS（P < 0.05）. Conclusion：Different functional mutation combinations revealed multiple sub-clones in myeloid malignancies with normal karyotype. The genomic landscape of AML patients was different from MDS patients.